1. Field of the Invention
The object of the present invention is mycobacterial proteins and microorganisms producing them.
It also relates to the use of these proteins in vaccines or for the detection of tuberculosis.
2. Discussion of the Background
Tuberculosis continues to be a public health problem throughout the world. The annual number of deaths directly related to tuberculosis is about 3 million and the number of new cases of tuberculosis is about 15 million. This number of deaths due to tuberculosis is high even for the developed countries; for example in France it is of the order of 1500 per year, a figure which is certainly underestimated by a factor of 2 or 3 if Roujeau's assessments of the differences between official figures and the results of systematic autopsies are taken into account. The recent increase in tuberculosis cases, or at least the leveling-off of the decrease in the frequency of this disease, must be considered in correlation with the development of the HIV/AIDS epidemic. In total, tuberculosis remains the leading infectious disease in terms of frequency in France and the developed countries, but above all in the developing countries for which it constitutes the principal source of human loss related to a single disease.
At present, a definite diagnosis made by the demonstration of cultivatable bacilli in a sample taken from the patient is only obtained in less than half the cases of tuberculosis. Even for pulmonary tuberculosis, which represents 80 to 90% of the tuberculosis cases, and which is the form of the disease for which the detection of the bacilli is the easiest, the examination of expectorations is only positive for less than half the cases.
The development of more sensitive techniques such as PCR (amplification by polymerase chain reaction), always comes up against the necessity for obtaining a sample. Women and children do not normally spit, and samples for infants frequently require relatively specialized medical intervention (for example ganglionic biopsy or sampling by lumbar puncture of the cephalorachidian fluid).
In other respects, inhibitions of the PCR reaction itself exist, of a type such that a sample may be unusable by this technique because of the impossibility of controlling its origins.
Finally, because of its limits of sensitivity (at the best of the order of 10.sup.4 to 10.sup.5 bacilli in the sample) the classic bacteriological diagnosis, microscopic examination and culture, requires that there has already been a relatively substantial development of bacilli and thus of the disease.
The detection of specific antibodies directed against Mycobacterium tuberculosis should thus be of assistance in the diagnosis of the common forms of the disease for which the detection of the bacilli themselves is difficult or impossible.
Successive generations of research workers have attempted to perfect a serological diagnostic technique for tuberculosis.
For a general review of studies carried out in this area, the application PCT WO-92/21758 may advantageously be referred to.
The techniques reported in the prior art are thus largely based on the preliminary isolation of proteins through their biochemical properties. It is not until after this isolation that the authors have tested the capacity of these proteins to detect those individuals affected by tuberculosis.
Application PCT WO-92/21758 describes a method for unambiguously selecting representative antigens of tubercular infection using serums originating from patients affected by tuberculosis or guinea-pigs immunized by live bacilli. This method, which is distinguished from the majority of the experiments described in the prior art, has led to the isolation of M. bovis proteins with molecular weights between 44.5 and 47.5 kD.
The seventeen amino acids of the N-terminal of one of these proteins were determined and are the following (SEQ ID NO: 5):
ALA-PRO-GLU-PRO-ALA-PRO-PRO-VAL-PRO-PRO-ALA-ALA-ALA-ALA-
1 2 3 4 5 6 7 8 9 10 11 12 13 14
PRO-PRO-ALA
15 16 17
The article by ROMAIN et al. (1993, Infection and immunity, 61, 742-750) recapitulates the substance of the results described in this international application. It more particularly describes a competitive ELISA assay using a rabbit polyclonal immune serum obtained by immunizing rabbits against the 45-47 kD protein complex described above.
In parallel, a gene library from Mycobacterium tuberculosis has been created by JACOBS et al. (1991, Methods Enzymol., 204, 537-557).
This library contains a large number of different clones.
A protein from another Mycobacteria species, M. leprae, has moreover been identified by WIELES et al. (1994, Infection and Immunity, 62, 252-258). This protein, named 43 L, has a molecular weight deduced from the nucleotide sequence of about 25.5 Da. Its N terminal has 47% homology with that of the 45-47 kDa protein complex identified in Mycobacterium bovis BCG, and whose 17 amino acid sequence is given above.
As stated above, there is a major interest in human medicine, as much from the therapeutic as the diagnostic point of view, in accurately identifying the proteins produced by the Mycobacteria and in particular by M. tuberculosis.
The problem which is in fact posed and is as yet unresolved lies in obtaining vaccines against a large number of diseases.
Another problem lies in the detection of diseases induced by the Mycobacteria, such as tuberculosis.
The applicant has thus pursued the determination of the sequence of a Mycobacterium tuberculosis protein, which is suspected of playing a major role in the immune response.
The applicant has demonstrated that the group of proteins corresponding to the 45-47 kD complex described above is coded by one and the same gene, and that the calculated molecular mass is different from the molecular mass estimated on polydcrylamide gel, because of its richness in proline.